1 Immunity to Mycobacterium tuberculosis (Mtb) has been the subject of detailed investigations for a number of 2 decades. Most studies have focused on classically restricted (MHC class Ia or II) T-cells, but recently, non- 3 classically restricted T-cells have become subject of increasing interest. Non-classical presentation molecules 4 (which include human CD1a,b,c,d; MR1; HLA-E and other HLA class Ib molecules) have been shown to 5 present Mtb antigens to human T-cells. HLA-E is a particularly interesting molecule, for a number of reasons: 6 first, there are only 2 coding variants described, which differ in only a single amino acid, located outside the 7 peptide binding groove, such that HLA-E essentially is a conserved, virtually monomorphic presentation 8 molecule; secondly, in contrast to HLA class Ia molecules, HLA-E is relatively resistant to HIV mediated 9 downregulation, such that antigen presentation should be maintained during HIV infection; thirdly, HLA-E is 10 present in Mtb containing intracellular compartments. Finally, we have recently shown that human CD8+ T- 11 cells recognizing Mtb peptides presented by HLA-E are present in the blood of Mtb infected individuals. 12 These cells have an unconventional phenotype and function: they had cytolytic properties, were able to 13 control intracellular outgrowth of Mtb, but unexpectedly produced mainly Th2 cytokines, including IL-4, IL-5 14 and IL-13, and efficiently helped B-cells through IL-4. 15 In the present project we propose an in depth characterization of these HLA-E restricted T-cells, both by 16 detailed characterization of available T-cell clones we have generated as well as by enumeration and 17 characterization of HLA-E restricted T-cells in (blood and BAL of) clinically relevant cohorts. The first part of 18 the project will characterize Mtb specific, HLA-E restricted T-cell clones, focusing first on their T-cell receptor 19 (TCR), including the requirements for peptide recognition and molecular structure of the TCR. In addition, the 20 clones will be employed to decipher the mechanism of intracellular Mtb growth inhibition, as this will be 21 important for understanding protective immunity towards Mtb. In the second part of the project, we will 22 enumerate HLA-E restricted T-cells in cohorts of Mtb infected individuals and individuals vaccinated with 23 BCG, both parenteral and aerosolized. These human studies will be bridged to parallel BCG vaccination 24 studies in non-human-primates, to allow validation of HLA-E restricted T-cell responses following vaccination 25 as well as following TB challenge. For all these studies unique collections of banked samples will be made 26 available by top edge collaborators (from UK, Italy and SA). Finally, tetramer (TM) positive cells will be sorted 27 to allow for RNA sequencing for full characterization of HLA-E restricted CD8+ T-cell responses. 28 All together these studies are expected to yield important new insights in the functional characteristics, clinical 29 relevance and vaccine kinetics of human Mtb specific, HLA-E restricted CD8+ T-cells. This will provide novel 30 insights into their potency as TB biomarkers and future TB vaccine targets.